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Virol JVirology Journal1743-422XBioMed Central London 1743-422X-2-51570308510.1186/1743-422X-2-5HypothesisThe Severe Acute Respiratory Syndrome (SARS)-coronavirus 3a protein may function as a modulator of the trafficking properties of the spike protein Tan Yee-Joo 1mcbtanyj@imcb.a-star.edu.sg1 Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, 138673 Singapore2005 10 2 2005 2 5 5 17 1 2005 10 2 2005 Copyright © 2005 Tan; licensee BioMed Central Ltd.2005Tan; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background
A recent publication reported that a tyrosine-dependent sorting signal, present in cytoplasmic tail of the spike protein of most coronaviruses, mediates the intracellular retention of the spike protein. This motif is missing from the spike protein of the severe acute respiratory syndrome-coronavirus (SARS-CoV), resulting in high level of surface expression of the spike protein when it is expressed on its own in vitro.

Presentation of the hypothesis
It has been shown that the severe acute respiratory syndrome-coronavirus genome contains open reading frames that encode for proteins with no homologue in other coronaviruses. One of them is the 3a protein, which is expressed during infection in vitro and in vivo. The 3a protein, which contains a tyrosine-dependent sorting signal in its cytoplasmic domain, is expressed on the cell surface and can undergo internalization. In addition, 3a can bind to the spike protein and through this interaction, it may be able to cause the spike protein to become internalized, resulting in a decrease in its surface expression.

Testing the hypothesis
The effects of 3a on the internalization of cell surface spike protein can be examined biochemically and the significance of the interplay between these two viral proteins during viral infection can be studied using reverse genetics methodology.

Implication of the hypothesis
If this hypothesis is proven, it will indicate that the severe acute respiratory syndrome-coronavirus modulates the surface expression of the spike protein via a different mechanism from other coronaviruses. The interaction between 3a and S, which are expressed from separate subgenomic RNA, would be important for controlling the trafficking properties of S. The cell surface expression of S in infected cells significantly impacts viral assembly, viral spread and viral pathogenesis. Modulation by this unique pathway could confer certain advantages during the replication of the severe acute respiratory syndrome-coronavirus.
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Background
The recent severe acute respiratory syndrome (SARS) epidemic, which affected over 30 countries, resulted in more than 8000 cases of infection and more than 800 fatalities (World Health Organization, ). A novel coronavirus was identified as the aetiological agent of SARS [1]. Analysis of the nucleotide sequence of this novel SARS coronavirus (SARS-CoV) showed that the viral genome is nearly 30 kb in length and contains 14 potential open reading frames (ORFs) [2-4]. These viral proteins can be broadly classified into 3 groups; (i) the replicase 1a/1b gene products which are important for viral replication, (ii) the structural proteins, spike (S), nucleocapsid (N), membrane (M) and envelope (E), which have homologues in all known coronaviruses, and are important for viral assembly, and (iii) the "accessory" proteins that are specifically encoded by SARS-CoV. Much progress have been made in characterizing these SARS-CoV proteins [5,6], but the molecular determinant for the severe clinical manifestations of SARS-CoV infection in contrast to the mild diseases caused by most coronaviruses, remains to be determined. In addition, the exact roles of "accessory" proteins of SARS-CoV are still poorly understood.

The subject of this hypothesis relate to the S protein and one of the "accessory" proteins, the SARS-CoV 3a protein. The S protein, which forms morphologically characteristic projections on the virion surface, mediates binding to cellular receptor and the fusion of viral and host membranes, both of these processes being critical for virus entry into host cells [7,8]. As such, S is known to be responsible for inducing host immune responses and virus neutralization by antibodies [9,10]. 3a (also termed ORF3 in [2] and [11], as X1 in [3], and as U274 in [12,13]) is the largest "accessory" protein of SARS-CoV, consisting of 274 amino acids and 3 putative transmembrane domains. Three groups independently reported the expression of 3a in SARS-CoV infected cells [13-15] and it was also detected in a SARS-CoV infected patient's lung specimen [14]. Antibodies against 3a were also found in convalescent patients [11,12,14].

This article hypotheses that the endocytotic properties of 3a allow it to modulate the surface expression of S and explores a functional significance for the interaction between S and 3a, which has been observed experimentally [13,15].

Presentation of the hypothesis
The cellular fate of the S protein has been well mapped [16,17]: S is cotranslationally glycosylated and oligomerized at the endoplasmic reticulum. Its N-linked high mannose side chains are trimmed, modified and become endoglycosidase H-resistant during the transportation to the Golgi apparatus. Only this fully-matured form of S can be assembled into virions and/or transported to the cell surface. The latter could cause cell-cell fusion and the formation of syncytia. Recently, Schwegmann-Wessels and co-worker reported that a novel sorting signal for intracellular localization is present in the S protein of most coronaviruses, but absent from SARS-CoV S [18]. Site-directed mutagenesis studies confirmed that a YxxΦ motif (where x is any amino acid and Φ is an amino acid with a bulky hydrophobic side chain) retains the S protein of TGEV intracellularly when it is expressed alone. On the other hand, SARS-CoV S is transported efficiently to the cell surface unless such a motif is introduced into its cytoplasmic tail by mutagenesis.

The YxxΦ motif has been implicated in directing protein localization to various intracellular compartments [19-21]. Furthermore, most YxxΦ motifs are capable of mediating rapid internalization from the plasma membrane into the endosomes. Interaction between the adaptor protein complex 2 (AP-2) with the YxxΦ motif present in the cytoplasmic domain of the internalizing protein concentrated the protein in clathrin-coated vesicle, which then budded from the plasma membrane resulting in internalization. However, it appears that the YxxΦ motif can also bind other adaptor protein complexes, like AP-1, 3 and 4, and the differential binding to the different adaptors will determine the pathway of a cargo protein containing a particular YxxΦ motif [21]. Coincidently, a YxxΦ motif in the cytoplasmic domain of 3a has previously been identified [13]. Furthermore, the juxtaposition of the YxxΦ motif and a ExD (diacidic) motif was found to be essential for the transport of 3a to the cell surface, consistent with the role of these motifs in the transportation of other proteins to the plasma membrane [22]. 3a on the cell surface can also undergo internalization [13].

Analyzing the experimental results present in these publications collectively, it is possible to postulate a functional role for the evolution of the SARS-CoV 3a protein. The SARS-CoV S protein lacks the YxxΦ motif but it can bind to the 3a protein which has internalization properties. In SARS-CoV infected cells, S is rapidly transported to the cell surface. But if 3a is expressed in the same cell, it is also transported to the cell surface where it can bind S. The interaction between 3a and S enables both proteins to become internalized, resulting in a decrease in the expression of S on the cell surface. Thus, this viral-viral interaction confers the functional role for the YxxΦ motif found in other coronaviruses to the SARS-CoV S. This hypothesis also implies that the precise mechanisms used by TGEV and SARS-CoV to reduce the expression of S are different although in both cases, the YxxΦ motifs will be crucial. In TGEV, the YxxΦ motif in S caused it to be retained intracellularly, while in SARS-CoV, S that is transported to the cell surface becomes internalized again after it interacts with the 3a protein.

Testing the hypothesis
Using mammalian cell culture system and biochemical methods, it will be possible to determine the exact effects of 3a on the trafficking properties of S. Mutagenesis studies can be used to map the protein domains that are important for the interaction between 3a and S and for the defining the manner by which 3a contributes to the reduction of cell surface expression of S. Given that a full-length infectious clone of SARS-CoV has been assembled [23], the use of reverse genetics would certainly reveal more about the interplay between 3a and S during SARS-CoV infection.

Implication of the hypothesis
This hypothesis, if proven, will indicate that the interaction between SARS-CoV-unique 3a protein and S results in a reduction of S on the cell surface through the endocytotic properties of 3a [13]. During SARS-CoV infection, expression of S on the cell surface of an infected cell mediates fusion with un-infected neighboring cells, leading to syncytium formation. It follows that reducing the cell surface expression of S will delay this cell-damaging effect and prevent the premature release of unassembled viral RNA. It may also enhance virus packaging as it appears that the assembly of coronavirus occurs intracellularly, probably in the intermediate compartments between the endoplasmic reticulum and Golgi apparatus [24]. Clearly, this has certain advantages for the virus at certain stages of its life cycle. In addition, a reduction in the cell surface expression of S may also help the infected cell evade the host defense system and reduce the production of anti-S neutralizing antibodies. Conversely, host or viral factors that disrupt the interaction between S and 3a would favor the expression of S on the cell surface and enhance cell-cell fusion, a process that is important for viral spreading.

Table 1 shows a comparison of the amino acid sequences of the cytoplasmic tails of the S protein of different coronaviruses, including SARS-CoV, which is distantly related to the established group 2 coronaviruses [25], as well as two recently identified novel human coronaviruses, HCoV-NL63 [26] and HCoV-HKU1 [27]. The YxxΦ motifs are clearly present in all group 1 coronaviruses and also in IBV, which belongs to group 3. However, no YxxΦ motif is present in SARS-CoV and MHV, both group 2 coronaviruses. In addition, there is a YGGR motif in the S protein of RtCoV and YxxH motifs in the S proteins of the other group 2 coronaviruses, BCoV, HEV and HCoV-HKU1. However, these motifs may not be able to function as signaling motifs because both R and H are not hydrophobic amino-acids. Therefore, HCoV-OC43 is the only one of these group 2 coronaviruses that encodes a S protein with a YxxΦ motif. It is still unclear how the localization of S is modulated in those viruses that lack YxxΦ motifs in the S proteins and further studies will be needed to understand the different signaling pathways that are important for regulating the trafficking properties of S. Indeed, the dilysine endoplasmic reticulum retrieval signal, which is a different type of sorting signal from the YxxΦ motif, in the cytoplasmic tail of IBV was reported to be important for intracellular retention of S [28].

Table 1 Amino acid sequences of the cytoplasmic tail of spike (S) proteins of coronaviruses are compared with the YxxΦ (where x is any amino acid and Φ is an amino acid with a bulky hydrophobic side chain) motifs found in SARS-CoV 3a protein and other cellular proteins that are known to undergo endocytosis.

Protein	Amino acid sequences in the cytoplasmic taila	
TGEV Sb	TM-CLGSCCHSICSRRQFENYEPIEKVHVH	
PRCoV Sb	TM-CLGSCCHSIFSRRQFENYEPIEKVHVH	
CCoV Sb	TM-CLGSCCHSICSRGQFESYEPIEKVHVH	
FCoV Sb	TM-CLGSCCHSICSRRQFENYEPIEKVHVH	
PEDV Sb	TM-CCGACFSGCCRGPRLQPYEAFEKVHVQ	
HCoV-229E Sb	TM-CFASSIRGCCESTKLPYYDVEKIHIQ	
HCoV-NL63 Sb	TM-CLTSSMRGCCDCGSTKLPYYEFEKVHVQ	
BCoV Sc	TM-ICGGCCDDYTGHQELVIKTSHDD	
HCoV-OC43 Sc	TM-KCGGCCDDYTGYQELVIKTSHDD	
HEV Sc	TM-KCGGCCDDYTGHQEFVIKTSHDD	
MHV Sc	TM-KKCGNCCDECGGHQDSIVIHNISSHED	
RtCoV Sc	TM-KCGNCCDEYGGRQAGIVIHNISSHED	
HCoV-HKU1 Sc	TM-KCHNCCDEYGGHHDFVIKTSHDD	
SARS-CoV Sc	TM-GACSCGSCCKFDEDDSEPVLKGVKLHYT	
IBV Sd	TM-KKSSYYTTFDNDVVTEQYRPKKSV	
SARS-CoV 3ae	TM-38aa-YNSVTDTIVVTEGD-101aa	
TfRe	19aa-YTRFSLARQVDGDNSHV-26aa-TM	
LDLR (proximal)e	TM-17aa-YQKTTEDEVHICH-20aa	
LDLR (distal)e	TM-34aa-YSYPSRQMVSLEDDVA	
CD-M6PRe	TM-34aa-YRGVGDDGLGEESEERDDHLLPM	
ASGPRe	MTKEYQDLQHLDNEES-24aa	
aSequences were obtained from National Center for Biotechnology Information (NCBI). Yxxx tetrapeptides are underlined and abbreviations used are: TM, transmembrane domain, aa, amino acids.

bS proteins of group 1 coronaviruses: TGEV, transmissible gastroenteritis virus (AJ271965); PRCoV, porcine respiratory coronavirus (Z24675); CCoV, canine coronavirus (D13096); FCoV, feline coronavirus (AY204704); PEDV, porcine epidemic diarrhea virus (AF353511); HCoV-229E, human coronavirus 229E (AF304460); HCoV-NL63, human coronavirus NL63(AY518894).

cS proteins of group 2 coronaviruses: BCoV, bovine coronavirus (AF220295), HCoV-OC43, human coronavirus OC43 (AY585228), HEV, porcine hemagglutinating encephalomyelitis virus (AY078417), MHV, murine hepatitis virus (AF201929), RtCoV, rat coronavirus (AF207551), HCoV-HKU1, human coronavirus HKU1 (AY597011), SARS-CoV, SARS coronavirus (AY283798).

dS protein of group 3 coronavirus: IBV, infectious bronchitis virus (M95169).

eSARS-CoV 3a protein (AY283798) and other cellular proteins that are known to undergo endocytosis. Abbreviations: TfR, transferrin receptor (P02786), LDLR, low-density lipoprotein receptor (P01130); CD-M6PR, cation-dependent mannose 6-phosphate receptor (P24668); ASGPR, asialoglycoprotein receptor (P07306).

It therefore appears that the cell surface expression of S protein of SARS-CoV can be reduced like that for other coronaviruses, but the mechanism may be different. The trafficking of SARS-CoV S may be mediated through 2 separate viral proteins, expressed from separate subgenomic RNA, and regulated by numerous complex cellular processes including the efficiency of transcription and translation, post-translation modification and stability of the viral proteins, as well as their interactions with host factors. Indeed, it is crucial to determine how this unique pathway benefits replication of the SARS-CoV. It is also interesting to note that sequence comparison of isolates from different clusters of infection showed that both S and 3a showed a positive selection during virus evolution [29,30], implying that these proteins play important roles in the virus life cycle and/or disease development and is consistent with the proposal that 3a has evolved to modulate the trafficking properties of the spike protein.

Competing interests
The author(s) declare that they have no competing interests.

Author's contributions
Yee-Joo Tan is responsible for the entire manuscript.

Acknowledgements
This work was supported by grants from the Agency for Science, Technology and Research (A*STAR), Singapore.
==== Refs
Drosten C Preiser W Gunther S Schmitz H Doerr HW  Severe acute respiratory syndrome: identification of the etiological agent Trends Mol Med 2003 9 325 327 12928032 10.1016/S1471-4914(03)00133-3 
Marra MA Jones SJ Astell CR. Holt RA Brooks-Wilson A Butterfield YS Khattra J Asano JK Barber SA Chan SY Cloutier A Coughlin SM Freeman D Girn N Griffith OL Leach SR Mayo M McDonald H Montgomery SB Pandoh PK Petrescu AS Robertson AG Schein JE Siddiqui A Smailus DE Stott JM Yang GS Plummer F Andonov A Artsob H Bastien N Bernard K Booth TF Bowness D Czub M Drebot M Fernando L Flick R Garbutt M Gray M Grolla A Jones S Feldmann H Meyers A Kabani A Li Y Normand S Stroher U Tipples GA Tyler S Vogrig R Ward D Watson B Brunham RC Krajden M Petric M Skowronski DM Upton C Roper RL  The Genome sequence of the SARS-associated coronavirus Science 2003 300 1399 1404 12730501 10.1126/science.1085953 
Rota PA Oberste MS Monroe SS Nix WA Campagnoli R Icenogle JP Penaranda S Bankamp B Maher K Chen MH Tong S Tamin A Lowe L Frace M DeRisi JL Chen Q Wang D Erdman DD Peret TC Burns C Ksiazek TG Rollin PE Sanchez A Liffick S Holloway B Limor J McCaustland K Olsen-Rasmussen M Fouchier R Gunther S Osterhaus AD Drosten C Pallansch MA Anderson LJ Bellini WJ  Characterization of a novel coronavirus associated with severe acute respiratory syndrome Science 2003 300 1394 1399 12730500 10.1126/science.1085952 
Thiel V Ivanov KA Putics A Hertzig T Schelle B Bayer S Weissbrich B Snijder EJ Rabenau H Doerr HW Gorbalenya AE Ziebuhr J  Mechanisms and enzymes involved in SARS coronavirus genome expression J Gen Virol 2003 84 2305 2315 12917450 10.1099/vir.0.19424-0 
Ziebuhr J  Molecular biology of severe acute respiratory syndrome coronavirus Curr Opin Microbiol 2004 7 412 419 15358261 10.1016/j.mib.2004.06.007 
Tan Y-J Lim SG Hong W  Characterization of viral proteins encoded by the SARS-Coronavirus genome Antiviral Research 2005 65 69 78 15708633 10.1016/j.antiviral.2004.10.001 
Cavanagh D  Siddell SG  The coronavirus surface glycoprotein protein The Coronaviridae 1995 New York: Plenum Press 73 113 
Gallagher TM Buchmeier MJ  Coronavirus spike proteins in viral entry and pathogenesis Virology 2001 279 371 374 11162792 10.1006/viro.2000.0757 
Holmes KV  SARS coronavirus: a new challenge for prevention and therapy J Clin Invest 2003 111 1605 1609 12782660 10.1172/JCI200318819 
Navas-Martin S Weiss SR  SARS: lessons learned from other coronaviruses Viral Immunol 2003 16 461 474 14733734 10.1089/088282403771926292 
Guo JP Petric M Campbell W McGeer PL  SARS corona virus peptides recognized by antibodies in the sera of convalescent cases Virology 2004 324 251 256 15207612 10.1016/j.virol.2004.04.017 
Tan Y-J Goh P-Y Fielding BC Shen S Chou C-F Fu J-L Leong HN Leo YS Ooi EE Ling AE Lim SG Hong W  Profile of antibody responses against SARS-Coronavirus recombinant proteins and their potential use as diagnostic markers Clin Diag Lab Immunol 2004 11 362 371 10.1128/CDLI.11.2.362-371.2004 
Tan Y-J. Teng E Shen S Tan THP Goh P-Y Fielding BC Ooi E-E Tan H-C Lim SG Hong W  A novel SARS coronavirus protein, U274, is transported to the cell surface and undergoes endocytosis J Virol 2004 78 6723 6734 15194747 10.1128/JVI.78.13.6723-6734.2004 
Yu C-J Chen Y-C Hsiao C-H Kuo T-C Chang SC Lu C-Y Wei W-C Lee C-H Huang L-M Chang M-F Ho H-N Lee FJS  Identification of a novel protein 3a from severe acute respiratory syndrome coronavirus FEBS Lett 2004 565 111 116 15135062 10.1016/j.febslet.2004.03.086 
Zeng R Yang RF Shi MD Jiang MR Xie YH Ruan HQ Jiang XS Shi L Zhou H Zhang L Wu XD Lin Y Ji YY Xiong L Jin Y Dai EH Wang XY Si BY Wang J Wang HX Wang CE Gan YH Li YC Cao JT Zuo JP Shan SF Xie E Chen SH Jiang ZQ Zhang X Wang Y Pei G Sun B Wu JR  Characterization of the 3a protein of SARS-associated coronavirus in infected vero E6 cells and SARS patients J Mol Biol 2004 341 271 279 15312778 10.1016/j.jmb.2004.06.016 
Parker MD Yoo D Cox GJ Babiuk LA  Primary structure of the S peplomer gene of bovine coronavirus and surface expression in insect cells J Gen Virol 1990 71 263 270 2155283 
Vennema H Heijnen L Zijderveld A Horzinek MC Spaan WJ  Intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly J Virol 1990 64 339 346 2403441 
Schwegmann-Wessels C Al-Falah M Escors D Wang Z Zimmer G Deng H Enjuanes L Naim HY Herrler G  A novel sorting signal for intracellular localization is present in the S protein of a porcine coronavirus but absent from severe acute respiratory syndrome-associated coronavirus J Biol Chem 2004 279 43661 4366 15304515 10.1074/jbc.M407233200 
Trowbridge IS Collawn JF Hopkins CR  Signal-dependent membrane protein trafficking in the endocytic pathway Ann Rev Cell Biol 1993 9 129 161 8280459 
Marks MS Ohno H Kirchhausen T Bonifacino JS  Protein sorting by tyrosine- based signals: adapting to the Ys and wherefores Trends Cell Biol 1997 7 124 128 17708922 10.1016/S0962-8924(96)10057-X 
Bonifacino JS Traub LM  Signals for sorting of transmembrane proteins to endosomes and lysosomes Annu Rev Biochem 2003 72 395 447 12651740 10.1146/annurev.biochem.72.121801.161800 
Bannykh SI Nishimura N Balch WE  Getting into the Golgi Trends Cell Biol 1998 8 21 25 9695803 10.1016/S0962-8924(97)01184-7 
Yount B Curtis KM Fritz EA Hensley LE Jahrling PB Prentice E Denison MR Geisbert TW Baric RS  Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus Proc Natl Acad Sci USA 2003 100 12995 13000 14569023 10.1073/pnas.1735582100 
Klumperman J Locker JK Meijer A Horzinek MC Geuze HJ Rottier PJ  Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding J Virol 1994 68 6523 6534 8083990 
Snijder EJ Bredenbeek PJ Dobbe JC Thiel V Ziebuhr J Poon LL Guan Y Rozanov M Spaan WJ Gorbalenya A  Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage J Mol Biol 2003 331 991 1004 12927536 10.1016/S0022-2836(03)00865-9 
van der Hoek L Pyrc K Jebbink MF Vermeulen-Oost W Berkhout RJ Wolthers KC Wertheim-van Dillen PM Kaandorp J Spaargaren J Berkhout B  Identification of a new human coronavirus Nat Med 2004 10 368 373 15034574 10.1038/nm1024 
Woo PC Lau SK Chu CM Chan KH Tsoi HW Huang Y Wong BH Poon RW Cai JJ Luk WK Poon LL Wong SS Guan Y Peiris JS Yuen KY  Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia J Virol 2005 79 884 895 15613317 10.1128/JVI.79.2.884-895.2005 
Lontok E Corse E Machamer CE  Intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site J Virol 2004 78 5913 5922 15140989 10.1128/JVI.78.11.5913-5922.2004 
Guan Y Peiris JS Zheng B Poon LL Chan KH Zeng FY Chan CW Chan MN Chen JD Chow KY Hon CC Hui KH Li J Li VY Wang Y Leung SW Yuen KY Leung FC  Molecular epidemiology of the novel coronavirus that causes severe acute respiratory syndrome Lancet 2004 363 99 104 14726162 10.1016/S0140-6736(03)15259-2 
Yeh SH Wang HY Tsai CY Kao CL Yang JY Liu HW Su IJ Tsai SF Chen DS Chen PJ National Taiwan University SARS Research Team  Characterization of severe acute respiratory syndrome coronavirus genomes in Taiwan: molecular epidemiology and genome evolution Proc Natl Acad Sci USA 2004 101 2542 2547 14983045 10.1073/pnas.0307904100

